The invention relates to the analysis of type, state or other distinguishing features of individual cells from body fluids, smears or tissues.
The invention comprises the steps of depositing the cells, with a minimum possible overlap, on a mass spectrometric sample support, determining the coordinates of the cells, coating the sample support with a layer of small crystals of a matrix substance, positioning the cells, inside a mass spectrometer, according to their known coordinates with a movement device into the position of the laser focus, acquiring mass spectra of the individual cells with ionization of the cell components by matrix assisted laser desorption, and using the mass spectra for an analysis of type, state or other distinguishing features of the cells.
Imaging mass spectrometry analysis of thin histologic sections or other flat samples with ionization of the molecules of interest using matrix assisted laser desorption (MALDI) has recently experienced an exceptional increase in popularity. Generally, the method is used to measure distributions of specific proteins which, either alone or in combination with other proteins, can serve as biomarkers for the visualization of various organs and, above all, for characterizing the stress or disease states of individual regions of the flat sample. No other method can at present characterize these stress or disease states as reliably and quickly. A method of this type is described in the patent application DE 10 2004 037 512.7 (D. Suckau et al., GB 2 418 773 A, U.S.-2006-0006315-A1).
In these processes, thin sections are typically applied to special specimen slides, the transparency of which permits microscopic observation and which feature a conductive layer so that later, in the mass spectrometer, they can provide a defined potential for the acceleration of the ions generated there.
The flat sample on the specimen slide must be covered with a layer of small matrix crystals in a special way to ensure that the proteins and also other substances of interest can be ionized effectively. A particularly favorable coating method is described in the patent application DE 10 2006 059 695.1 (M. Schürenberg, GB 2 446 251 A, US 2008/0142703 A1). This fine spraying or misting method is optically controlled, thereby achieving a dense, reproducible coverage with a layer of matrix crystals between 20 and 50 micrometers thick. Protein molecules, in particular, are drawn out of the sample to the surface of the layer. In combination with special laser beam profiles, the matrix layer surprisingly, and contrary to what had previously been believed, demonstrates a very high sensitivity, so that the most important proteins of even very small regions of the thin histologic section can be analyzed. The conventional understanding was that only one analyte ion would be formed from 10,000 analyte molecules. However, for reasons that have not yet been understood, the yield of protein ions from the layer of fine matrix crystals appears to be greater than this by a factor of at least 100, and possibly 1000, when special laser beam profiles are used.
When specially shaped laser beam profiles, such as those presented in the patent application DE 10 2004 044 196 A1 (A. Hase et al., GB 2 421 352 A, US 7,235,781 B2) are used, the analysis of the proteins can be restricted to regions with a diameter of only about five micrometers. The laser beam profile consists primarily of one or more laser beam points, each with a diameter of only five micrometers or less. Due to slight lateral diffusion when the matrix layer is applied, the spatial resolution when measuring the distribution of molecules in the flat samples is usually about 20 micrometers, which is perfectly adequate for the majority of applications.
To obtain a good quality measurement, with high sensitivity and good precision in the measurement of concentration, it is not sufficient, however, to record a single spectrum based on a single laser pulse. Rather, between 20 and 500 individual spectra are added to form a sum spectrum. When the term “mass spectrum” is used below, it is this sum spectrum that is always meant. If the spatial resolution is fully exploited by taking the measurements with a 20 micrometer grid spacing, this means that 250,000 mass spectra, composed of many millions of individual spectra, will be recorded for each square centimeter of thin section. If the recording speed is one mass spectrum per second on the basis of, for instance, 200 individual spectra recorded at 200 Hz, this process will take about 70 hours per square centimeter.
Of course, lower spatial resolutions can also be chosen; for cross-sections taken through the bodies of, for instance, mice or rats, grid spacings of between 200 and 500 micrometers permit a very good distribution of analyte substances across the individual organs and intermediate spaces to be measured. Here only 2500 or 400 mass spectra respectively need to be recorded per square centimeter; these may nevertheless still comprise between a hundred thousand and a million individual spectra. In this case again, the ability to record the individual spectra at a high frequency, preferably more than 1000 individual spectra per second, is desirable. However, these individual spectra must not be taken from a single point to avoid overheating the matrix layer at this site. It is therefore expedient to continuously vary the recording coordinates and, in particularly critical cases, to lower the recording rate down to, for instance, only 200 individual spectra per second.
It is, however, not just the mass spectrometric analysis of the state of tissues from parts of thin histologic sections that is of interest, but also the analysis of individual cells from smears, body fluids or tissues. The analyses may be aimed at determining the type of cells, or may be oriented toward the stress, disease or infection of the individual cells. Even the simple determination of distinguishing features is interesting, and the reasons for the distinguishing features do not even have to be known.
For such an analysis, the cells, if they are not already distributed in body fluids, must be dispersed, separated from one another, in a liquid. Equipment is commercially available specifically for preparing the cells from liquids on specimen slides. Here, the cells are applied to a small region of the specimen slide, for instance one square centimeter, by gentle centrifuging; they are pressed flat without being damaged, and occupy a space with a diameter of about 20 micrometers. Cells from tissues such as bone marrow can also be distributed in liquids, and then applied to specimen slides, using special procedures. If the number of cells in the liquid is small enough, there will be very few overlaps, and the medical professional will be able to observe the cells individually under a microscope. A “small enough number” of cells here means from a few hundred up to a maximum of about 10,000 cells per square centimeter. The optimum for the lowest possible percentage of overlaps is around 3000 cells per square centimeter.
The purpose of such an analysis is often to determine the presence of a few abnormal cells, tumor cells for instance, among a large number of normal cells; this is a laborious and very tiring task if the medical professional has to examine the cells visually. For many of these cases, staining methods are either not known or do often not provide very high contrast; visual detection of tumor cells is affected by a large number of subjective influences and it is hard to achieve an objective analysis. Therefore, automatable methods for this task are required.
Areas of tissue with abnormal cells, tumor cells for instance, in thin sections can, in principle, be recognized as such on the basis of their mass spectra, although these tissue regions are usually mixed with a large proportion, often up to 80%, of healthy cells. An obvious solution is to coat the specimen slide, to which the cells have been applied, with matrix material, in the same way as thin sections, and then to scan them in a mass spectrometer on a grid pattern in order to obtain mass spectra of the individual cells. If all the individual cells, without exception, are to be analyzed, the grid spacing must be dense, having a pitch of at most 20 micrometers. On an area of one square centimeter, this leads to the number, as mentioned above, of 250,000 mass spectra, incorporating millions of individual spectra, and to the time, also mentioned above, of 70 hours, even though there may only be about 1000 to 10,000 cells on the surface. The vast majority of the mass spectra are empty.
Methods of this sort are only possible if solid-state lasers are used. The nitrogen lasers mostly used until now have a life time of only about one million laser pulses. Solid-state lasers have a considerably longer life time, but require special beam shaping measures, which can, however, be designed in a way that is advantageous to the analysis. Mass spectrometers that operate with solid-state lasers are already commercially available.
When referring here to the “state of the cells”, this should be understood in the sense of a stress, a pathologic change, an infection or other change from a normal metabolic state of the same type of cell. As has already been explained, tumor cells are of particular significance to this method; tumorous tissue can be clearly distinguished from healthy tissue by mass spectrometry. In general terms, it must be possible to recognize the state from the pattern of substance concentrations that can be detected in the cell by mass spectrometry. The substances may be peptides or proteins that are under- or overexpressed, so creating a characteristic pattern. They may, however, also be post-translational modifications of proteins or decomposition products (metabolites), or accumulations of other substances, such as lipids in the tissue.
The objective of the invention is to analyze type and state of individual cells with a maximum possible degree of automation.